肺腺癌多药耐药细胞特异表达基因的克隆与鉴定  

Suppression Subtractive Hybridization Identified Genes Differentially Expressed in a Multidrug Resistance Cell Line of Human Lung Adenocarcinoma

作　　者：陈杰[1] 钱桂生[1] 黄桂君[1] 熊玮[1] 李靖[1] 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所,重庆400037

出　　处：《癌症》2001年第4期348-353,共6页Chinese Journal of Cancer

基　　金：重庆市卫生局医学科研项目!(00- 2001);第三军医大学疾病基因项目

摘　　要：目的：克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法：将肺腺癌多药耐药细胞（ SPC- A- 1/CDDP）作为实验方，肺腺癌细胞（ SPC- A- 1）作为对照方，应用抑制消减杂交技术，构建实验方特异表达 cDNA消减文库；用斑点杂交法初步筛选 cDNA消减文库后，将获得的阳性克隆进行测序和同源性分析（ Genbank），对新的 cDNA序列进行 Northern blot杂交验证。结果：建立了一个肺腺癌多药耐药细胞（ SPC- A- 1/CDDP）特异表达 cDNA消减文库，斑点杂交法初步筛选显示 23个克隆中有 SPC- A- 1/CDDP特异表达 cDNA片断，测序和同源性分析表明 2个 cDNA片断为新序列，其余 cDNA片断与已知基因有 93％～ 100％的同源性， Northern blot杂交结果表明 2个新的 cDNA片断来自 SPC- A- 1/CDDP细胞。结论： 2个新的 cDNA序列可能为未知肺腺癌多药耐药相关基因序列；抑制消减杂交法是克隆特异表达基因的有效方法。Objective: The aim of this study was to clone and screen multidrug resistance related gene of human adenocarcinoma cell. Methods: The suppression subtractive hybridization (SSH) was performed on human adenocarcinoma multidrug resistance cell line (SPC- A- 1/CDDP, as tester) and human adenocarcinoma cell line (SPC- A- 1, as driver). After the subtracted cDNA library being constructed, the dot blots was used to screen the subtracted cDNA library with forward and reverse- subtracted cDNA probes. The differentially expressed cDNA fragments in SPC- A- 1/CDDP was sequenced and analyzed through Genbank with Blast search. The novel cDNA sequences were analyzed by Northern blots. Results: A high quality subtracted cDNA library was constructed. Twenty- three differentially expressed cDNA fragments in SPC- A- 1/CDDP were identified. Two of them were novel cDNA sequences and the others had 93％－ 100％ homology with the known genes respectively. Northern blots indicated the novel cDNA sequences only expressed in SPC- A- 1/CDDP cell. Conclusion: The novel cDNA sequences might be multidrug resistance related genes in human lung adenocarcinoma. SSH is a powerful technique to identify differentially expressed genes.

关 键 词：人肺腺癌细胞 多药耐药相关基因 抑制消减杂交 克隆 

分 类 号：R734.2[医药卫生—肿瘤] Q785[医药卫生—临床医学]